The objective of this research is to accurately define the sequence of molecular and enzymological events that occur during the replication of mammalian chromosomes. We are using simian virus 40 chromosomes as a suitable and relatively simple model of a single mammalian replicon. We have developed several subcellular systems that can faithfully continue both DNA replication and chromatin assembly in vitro in order to study the replication of normal endogenous viral nucleoproteins rather than purified DNA added to cell lysates and purified proteins. Our task is to fractionate these subcellular systems in an effort to isolate and characterize cellular and viral factors that are involved in the replication of mammalian chromosomes. We are now asking four specific questions. Are RNA primers required for the initiation of Okazaki fragments, and, if so, how are they synthesized and excised? How does DNA polymerase alpha specifically interact with native replicating chromosomes to allow the synthesis and completion of Okazaki fragments? What is the relationship between the initiation of Okazaki fragments and the phasing of pre-fork nucleosomes? What is the mechanism of separation of progeny DNA molecules and termination of DNA synthesis? We hope to combine the answers to these questions with data on the structure of replicating chromosomes in an effort to provide a comprehensive view of replicating chromosomes.